Abstract - AAV vectors are exceptionally efficient for gene delivery to the brain by direct intraparenchymal infusion, where they mediate continuous transgene expression perhaps for the lifetime of the experimental animal. These exceptional properties of existing AAV vectors are the basis for ongoing or planned clinical trials for neurological diseases where focal gene delivery is therapeutically effective. However development of gene therapy approaches for many other neurological diseases will require global gene delivery to the CNS. Here we will use in vivo selection of an AAV capsid library and molecular grafting to develop new CNS- targeted AAV vectors with such capability. In the first approach we will use in vivo selection of an AAV capsid library with a diversity of 5x109 clones generated by DNA shuffling of AAV1, 2, 5, 8, 9, and rh10 Cap genes, to identify new brain- and spinal cord-tropic AAV capsids after intravenous or ICV delivery in adult animals. The library will be infused via the tail vein or the cerebral lateral ventricles of adult mice and one month later we will isolate DNA from the brain and spinal cord for PCR amplification of tissue-resident AAV Cap genes. These will be cloned back into the original library plasmid backbone and used to produce more AAV virions for subsequent rounds of in vivo selection. We will sequence 10 AAV Cap genes per round of selection. Once we determine convergence of tissue resident AAV Cap genes for each target, we will prepare recombinant AAV vectors encoding firefly luciferase (Fluc) using the newly selected capsids and infuse them into adult animals to determine their biodistribution. We will use bioluminescence imaging to assess distribution and kinetics of gene expression followed by biochemical quantification of Fluc activity in different organs and histological assessment of transduced cell distribution in brain and spinal cord. In the second approach we will combine AAV vectors with different proteins and peptides previously shown to be highly efficient in ferrying liposomes, enzymes, and siRNAs across the BBB after intravascular infusion. For this we will use chimeric AAV capsids carrying a 14 amino acid biotin acceptor peptide that allows for incorporation of biotin into the AAV capsid during packaging. As brain-targeting molecules we will use Streptavidin fused to a single-chain antibody specific for the mouse Transferrin receptor (TfR), or peptides derived from human ApoB-100, or Rabies virus glycoprotein. The biodistribution of the new AAV-molecular conjugates after intravenous infusion will be assessed as above. The therapeutic efficacy of all new CNS-targeted AAV vectors will be tested in a mouse model of GM1-gangliosidosis, which is a lysosomal storage disease that severely affects the CNS. We expect this new generation of CNS-targeted AAV vectors to foster the development of highly effective gene therapy approaches to treat many childhood, adult, and geriatric neurological diseases currently beyond the reach of modern medicine PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to develop a new generation of AAV vectors capable of targeting the brain via the vasculature. Achieving global gene delivery to the adult CNS remains the 'holy grail'of neural gene therapy, and it is likely necessary to develop effective therapies for many neurological diseases. Here we will investigate two approaches to develop new CNS-targeted AAV vectors: 1) In vivo selection of an AAV capsid library via intravascular and intracerebroventricular delivery in adult animals;2) AAV-molecular conjugates incorporating brain-tropic proteins and peptides. This new generation of AAV vectors may provide the means to develop highly effective gene therapy approaches to treat many childhood, adult, and geriatric neurological diseases currently beyond the reach of modern medicine. The NIDA/NIMH/NINDS EUREKA applications were reviewed differently from more traditional NIH grant mechanisms. Specifically, the review process consisted of two phases. During the first (i.e., electronic) phase a selected panel of reviewers were given the following guidelines by which to assess the applications. They were asked to determine whether they: Strongly Agree, Moderately Agree, Neither Agree nor Disagree, Moderately Disagree, or Strongly Disagree with these descriptions. Their ratings and any additional comments are below. These initial ratings also provided the basis for the review panel to determine whether an application would be discussed during an in person meeting. Because of the very stringent review criteria and limited pool of funds set aside for this program, the review panel chose only to discuss applications that garnered the most enthusiasm. The Resume and Summary of the Discussion above summarizes opinions of the person meeting and forms the basis of the final score. Significance: This study addresses an important problem and the outcome of the proposed studies will drive the field. The potential impact of the proposed research is exceptional, in terms of the magnitude of the impact and the size of the community affected. Innovation: The project is highly original and exceptionally innovative and seriously challenges existing paradigms or clinical practice. The project addresses a major barrier to progress in the field or it develops or employs exceptionally novel concepts, approaches, methodologies, tools, or technologies. Approach: The logic of the approach is sufficiently compelling despite the lack of experimental detail. The conceptual (or clinical) framework, design, methods, and analyses are adequately developed, well integrated and reasoned, and are appropriate for the aims of the project. The applicant acknowledges potential problem areas and considers alternative tactics. The information in the timeline inspires confidence that the PI will be able to document progress in each year of the award and either complete the project or demonstrate conclusively that it cannot be completed, despite good-faith efforts, during the term of the award. The requested duration of the award is appropriate for the proposed research. Investigators: The PD/PI(s) and other key personnel are appropriately trained and well-suited to carry out this work. Past achievements of the PI(s) suggest that the investigator(s) is/are exceptionally innovative and likely to make paradigm-shifting, high-impact discoveries. If the PI does not have a history of doing exceptionally innovative, high-impact research, the logic of the experimental plan suggests that there is at least some likelihood of success. The project is high priority for the PI(s), as indicated by the person-months of effort that the PI(s) will devote to it. For applications designating multiple PDs/PIs, the leadership plan, including the designated roles and responsibilities, governance, and organizational structure, are consistent with and justified by the aims of the project and the expertise of each of the PDs/PIs. Environment: The scientific environment(s), in which the work will be performed, contributes to the probability of success. The proposed studies benefit from unique features of the scientific environment, subject populations, or employ useful collaborative arrangements. There is evidence of institutional support.